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Int. J. Biosci. 2020 International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 17, No. 5, p. 176-183, 2020 RESEARCH PAPER OPEN ACCESS Extraction of stress hormones by fecal sampling of big cats, ungulates and mammals in captivity and wild by using enzyme linked immunoabsorbant assay (ELISA) Jahanzeb Sarwer1, Saqafat Ahmed1, Maryam Khan1, Sikander Hayat1, Bushra Nisar Khan2, Arif Malik1, Saba Shamim1* 1Institute of Molecular Biology and Biotechnology, The University of Lahore, Defence Road Campus, Lahore, Pakistan 2Department of Zoology, University of the Punjab, New Campus, Lahore-54590, Pakistan Key words: Immunoassay, estrogen, FGC, metabolite, ELISA. http://dx.doi.org/10.12692/ijb/17.5.176-183 Article published on November 12, 2020 Abstract The determination of fecal cortisol and steroids via immunoassay and extraction techniques is a widely accepted and used phenomena with respect to both captive and field study for the provision of the estimation of the regulating concentration of hormones in animals, which was achieved through non-invasive procedures. The reposition of fecal samples is a significant matter of concern due to the metabolism of fecal steroids by bacteria present in the feces of animals only after a few hours of deposition. In this study, the estimation of fecal hormones like estrogen (fE) and glucocorticoid (fGC) metabolites was carried out in big cats of the wild in captivity, such as lions, tigers, African lions, puma, jaguar, few ungulates, mouflon sheep, deer, chinkara, zebra and Punjab urial. The fecal samples (n=106) were collected from these wild animals and were treated with methanol to curb the metabolism of fecal hormones by bacteria. The expression of stress hormone levels in different animals as obtained by ELISA is given as 0.644±0.03 for Punjab urial followed by 0.619±0.02 for deer, 0.614±0.05 for Chinkara, 0.606±0.01 for tiger and Puma, 0.579±0.02 for lion and 0.061±0.04 for Jaguar. Much stress hormone was observed in Punjab urial whereas Jaguar was least affected by the stress. It showed that animals living in small and noisy environment are more affected by any disturbance in their surroundings as compared to the animals living in quiet and less noisy environment. * Corresponding Author: Saba Shamim [email protected] 176 Sarwer et al. Int. J. Biosci. 2020 Introduction The utility of minimally invasive sample media as The animals have been kept in zoos for centuries for biomarkers of stress responses depends on the degree entertainment, conservation, education and research to which the corticosteroid content of the sample purposes. To provide these animals with an represents adrenocortical activity. Commonly, this environment as close as possible to the wild, is very involves comparisons between corticosteroid essential as it stimulate animals to display natural concentrations in blood plasma with concentrations behavior and stay stress free (Schildkraut, 2016). in the alternative sample media (Cook, 2012). When an animal is in stress, it shows many Measuring fecal cortisol metabolites as an indicator of physiological changes or response. These changes can adrenocortical activity in animals, offers the be of many forms like, increase in heartbeat, elevated advantage of a simple sampling technique that will stress hormones (cortisol) circulation, increase in not interfere with the results of the study and enables metabolic rate and dehydration in severe cases of heat even long-term and as well as longitudinal studies. exposure (Bourne and Cunningham, 2019). The Thus, such methods will be a valuable tool in a variety environmental disturbances can activate or release of research fields such as animal welfare (handling, the stress hormones (glucocorticoids). The housing and transportation) and also in ethological glucocorticoid hormones are important to understand and environmental studies (Möstl and Palme, 2002). how animals deal with perturbations in their environment and they are related to an individual Materials and methods wellbeing (Dantzer et al., 2014). The glucocorticoids Samples collection concentration determined from biological matrices, A total of 107 animal feces samples of various wild big such as blood, saliva, urine or feces can act as a cats, ungulates, and some other mammals were potential indicator for level of stress (Webster et al., collected from Lahore zoo, Bahawalpur zoo, Lahore 2018). The difficulties in obtaining blood samples and safari zoo and Islamabad zoo. The environmental the recognition of the stressor effect of blood conditions of the animals and collection were noted. sampling are primary drivers for the use of non- invasive sample media (Claxton, 2011). The blood Samples preservation sample collection for measuring the glucocorticoids The samples were preserved by treating them with can bias the stress results in case of captive animals. methanol. The 2 grams of sample was taken and The non-invasive methods of measuring stress incubated at 60 °C for 15 minutes. After incubation, hormones are important especially for free roaming the samples were dried. They were crushed and 1 species as capturing of an animal can itself cause gram was added to 20 ml methanol. stress (Behringer and Deschner, 2017). ELISA performance The front‐line hormones to overcome the stress are The ELISA kit used in this study was comprised of the glucocorticoids and catecholamines (Grimmett et al., following items: ELISA well plate coated with cortisol 2008). When glucocorticoids hormone secretes in the with MAb, cortisols standards vials (0.5 ml), Enzyme blood stream, it is metabolized in the kidneys and conjugate 20X (1.2 ml), TMB substrate (12 ml), Stop liver and then excrete through urine and feces solution (12 ml), 20X wash concentrate (25 ml) and (DeRango et al., 2019). In most sample media, Assay Diluent (24 ml). The experiment was measurement of a specific corticosteroid is a performed in two batches. requirement depending on the species, e.g. cortisol in most mammals, or corticosterone in birds. However, Batch-I in samples involving products of excretion, In the first phase, the number of samples were 49 and methodologies that measure a broad range of numbers of standards were 7 which were provided structurally related compounds are probably optimal. with the ELISA Kit. A total prepared conjugate 177 Sarwer et al. Int. J. Biosci. 2020 solution for 56 samples was diluted with distilled conjugate solution was diluted with the distilled water water in 1:21 ratio. For 56 samples, 7 wells were set on (1:21). For 64 samples, 8 wells were set on a holding a holding stand. Every strip contained 8 wells in a stand. For each single well, 128 µl of enzyme- single column. For each well, 112 µl of enzyme- conjugate solution was prepared. The total conjugate conjugate solution was prepared. The total conjugate solution prepared for 64 samples were 12,800 µl or solution prepared for 56 samples was 11,200 µl. 200 µl for a single well. According to the procedure, Hence by dividing 11200 µl with total 56 number of 640 µl wash buffer (1X) was prepared for the required samples including standards, a value of 200 µl came samples. Prior to the assay process, the reagents were for a single well. The 560 µl Wash buffer (1X) was allowed to stand at room temperature for some time. prepared for the required samples. Prior to assay They were gently mixed before use. Eight strips of process, the reagents were allowed to stand at room coated wells were fixed in stand holder. The 200 µl of temperature for some time. They were gently mixed enzyme-conjugate solution was added to each well. before use. Seven strips of coated wells were fixed in a Afterward 25 µl of cortisol standard was added to the stand holder. The 200 µl of enzyme-conjugate first 6 wells using micro pipette. The solution was solution was added to each well. Afterward 25 µl of gently mixed with the help of pipette for 10 second. cortisol standard was added to the first 7 wells using a The 25 µl of sample was added in the remaining wells micropipette. The solution was gently mixed with the with the help of pipette and gently mixed. The ELISA help of pipette for 10 second. The 25 µl of sample was plate was left for incubation at room temperature for added in the remaining wells with the help of pipette an hour. After incubation the plate was washed with and gently mixed. The ELISA plate was left for 1X wash buffer (300 µl) and blotted on absorbent incubation at room temperature for an hour. After paper. TMB substrate (100 µl) was added in all plates incubation, the plate was washed with 1X wash buffer and incubated for 15 minutes at room temperature. (300 µl) and blotted on an absorbent paper. TMB Stop solution (50 µl) was added to all wells. The substrate (100 µl) was added in all plates and solution was mixed gently. Within 20 minutes of incubated for 15 minutes at room temperature. Stop adding the stop solution, the absorbance was read on solution (50 µl) was added to all wells. The solution ELISA reader. was mixed gently. Within 20 minutes of adding the stop solution, the absorbance was read on ELISA Results reader. The fecal samples (106) were collected from different zoos; Lahore zoo, Bahawalpur zoo, Lahore safari zoo Batch-II and Islamabad zoo. Maximum fecal samples were In the second phase, the number of samples were 58 collected from lions followed by tiger and deer. Only and the numbers of standards were 6 as provided two samples were collected for jaguar and zebra with the ELISA Kit. Hence the total prepared (Table 1). Table 1. Collection of fecal samples from different captivities/ zoos.